pubs.acs.org/Biochemistry Published on Web 03/12/2010 r 2010 American Chemical Society

3336 Biochemistry 2010, 49, 3336–3342

DOI: 10.1021/bi100176a

Circular Dichroism and UV Resonance Raman Study of the Impact of Alcohols on theGibbs Free Energy Landscape of an R-Helical Peptide†

Kan Xiong and Sanford A. Asher*

Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Received February 4, 2010; Revised Manuscript Received March 11, 2010

ABSTRACT: We used CD and UV resonance Raman spectroscopy to study the impact of alcohols on theconformational equilibria and relative Gibbs free energy landscapes along the RamachandranΨ-coordinateof a mainly poly-Ala peptide, AP with an AAAAA(AAARA)3A sequence. 2,2,2-Trifluoroethanol (TFE)most stabilizes the R-helix-like conformations, followed by ethanol, methanol, and pure water. The π-bulgeconformation is stabilized more than the R-helix, while the 310-helix is destabilized due to the alcohol-increased hydrophobicity. Turns are also stabilized by alcohols. We also found that while TFE induces moreR-helices, it favors multiple, shorter helix segments.

Protein (peptide) folding depends on both its primary sequenceand its solvent environment. Addition of alcohol to an aqueoussolution changes the hydration of the protein (peptide). Theresulting conformational changes can be used as a valuable toolfor probing protein (peptide)-water interactions (1-7). It isimportant to realize that despite intensive investigations over theyears, the mechanism(s) by which alcohols perturb proteinconformation is still poorly understood (8-18).

In this work, we used CD and UV resonance Raman (UVRR)spectroscopy to study the impact of alcohols on the conforma-tional equilibria and relative Gibbs free energy landscapes alongthe RamanchandranΨ-coordinate of a mainly poly-Ala peptide,AP with an AAAAA(AAARA)3A sequence. We find that theR-helix and π-bulge conformations are most stabilized by 2,2,2-trifluoroethanol (TFE), followed by ethanol, methanol, andpure water. Turn conformations are also stabilized. However,310-helices are destabilized. We also find that TFE induces anincreased abundance of R-helices. However, the average R-helixlength is decreased.

EXPERIMENTAL PROCEDURES

The 21-residue peptide AP with an AAAAA(AAARA)3Asequence was purchased from AnaSpec Inc. (>95% purity).Absolute methanol was purchased from J. T. Baker. Absoluteethanol was purchased from Pharmco. 2,2,2-Trifluoroethanol(TFE, 99.8% purity) was purchased from Acros. The pH 7solution samples contain 1 mg/mL AP and 0.05 M NaClO4.

The CD spectra were recorded by using a Jasco-715 spectro-polarimeter, by using a 0.02 cm path length cuvette.We co-addedfive individual CD spectra.

The UV resonance Raman (UVRR) apparatus was describedin detail by Bykov et al. (19). Briefly, 204 nm UV light (1 mWaverage power, 100 μm diameter spot) was obtained by mixingthe third harmonic with the fundamental (816 nm wavelength,1 kHz repetition rate, 0.6 W average power, 25-40 ns pulse

width) of a tunable Ti:Sapphire laser system from PhotonicsIndustries. The sample was circulated in a free surface, tempera-ture-controlled stream.A 180� sampling backscattering geometrywas used. The collected light was dispersed by a double mono-chromator onto a back-thinned CCD camera (Princeton Instru-ments Spec 10 System, 1.5 cm-1 resolution with a 100 μm slitwidth). We used 5 min accumulation times, and four accumula-tions were co-added. The 732 and 1379.5 cm-1 bands of Teflonwere utilized to calibrate the frequencies. The frequencies arereproducible to less than 1 cm-1. Raman spectrawere normalizedto the peak height of the 932 cm-1 ClO4

- band. No Ramansaturation occurs at these low excitation powers.

RESULTS

CD Measurements. Figure 1 shows the temperature depen-dence of the CD spectra of AP in pure water. The low-tempera-ture CD spectra show two troughs at 222 and 206 nm that arecharacteristic of R-helix conformations (20). As the temperatureincreases, the ellipticity at 222 nm, θ222, becomes less negative,indicating R-helix melting. The isosbestic point at 202 nmindicates that the melting behavior appears spectroscopically asa “two-state” process. Previous work by our group demonstratedthat theAPR-helix conformationmelts to a dominantly PPII-likeconformation (21).

Panels a and b of Figure 2 show the temperature dependenceof the mean residue ellipticity at 222 nm, θ222, of AP in purewater and in the presence of different alcohols. Alcohols increasethe R-helix content. At 20 �C and 25% alcohol by volume,TFE most stabilizes the R-helix, followed closely by ethanol andthen methanol, consistent with previous studies (8, 12, 18).At 50% (v/v) alcohol, ethanol is the most R-helix-stabilizing,followed by TFE and then methanol. As the alcohol con-centration increases from 25 to 50%, θ222 decreases in methanoland ethanol but changes little in TFE (Figure 2c). Previousstudies also showed that TFE does not appear in CD measure-ments to induce additional R-helix concentrations above 25%(v/v) (11).UVRR Measurements. The 204 nm UV Raman spectra

(UVRS) of AP in pure water (Figure 3) show mainly the amide

†The work was supported by National Institutes of Health GrantsGM8RO1EB002053 and 1RO1EB009089.*To whom correspondence should be addressed. Phone: (412) 624-

8570. Fax: (412) 624-0588. E-mail: asher@pitt.edu.

Article Biochemistry, Vol. 49, No. 15, 2010 3337

RR bands. In contrast, the 204 nm UVRS of AP in 50%methanol (Figure 3) show methanol Raman bands which wenumerically removed in theFigure 4UVRS.The resulting spectrashow the temperature dependence of the 204 nmUVRS of AP in50% methanol. As the temperature increases, the AmI bandupshifts from 1652 to 1658 cm-1 while theAmII band downshiftsfrom 1556 to 1552 cm-1 (22). Previous work (23) indicates thatwater hydrogen bonding to the peptide bond (PB) CdO siteincreases the CdO bond length and, thus, downshifts the AmIband, while water hydrogen bonding to the PBN-H site upshiftsthe AmII band. TheAmI (AmII) band in pure water (Figure 4) isupshifted (downshifted) relative to that in 50% methanol,indicating less CdO (N-H) hydrogen bonding in an alcoholsolution (23). The CR-H doublet (∼1372 and ∼1393 cm-1)frequency does not shift as the temperature increases but itsintensity increases.

The CR-H doublet intensity only slightly increases from 2 to20 �C, indicating little R-helix melting (24). Significant intensitychanges observed from 20 to 40 �C indicate extensive R-helixmelting. The AmIII3 band downshifts from ∼1264 cm-1 at 2 �Cto ∼1259 cm-1 at 40 �C while its intensity increases (22). UVRSof AP in other alcohols (not shown) show very similar R-helixmelting behaviors.

To calculate the R-helical fractions, we subtracted appropriateamounts of the temperature-dependent PPII-like conformationbasis spectra (22) from the measured and digitally smoothedUVRS of AP to minimize the CR-H region intensity in thedifference spectra. The basis spectral intensities subtracted aredirectly proportional to the concentrations of the PPII-likeconformation at each temperature. The resulting difference spec-tra appear to be mainly R-helix-like. Figure 5 shows UVRR-calculated fractions of R-helix-like conformation of AP in purewater and in 50% (v/v) alcohol. The R-helix-like conforma-tions are dramatically stabilized in alcohol and melt little asthe temperature increases. TFE stabilizes the R-helix-like con-formations the most, followed by ethanol, and then methanol,as previously observed (8, 12, 18). The R-helix-like conforma-tion melting curves in ethanol and in methanol are essentiallyidentical. These conclusions obviously differ from the CD con-clusions.

Figure 6 shows the calculated R-helix-like spectra of AP in

pure water and in 50% (v/v) methanol. The AmIII3 band in

pure water shows a peak at ∼1258 cm-1 with a shoulder at

∼1280 cm-1 and another shoulder at∼1240 cm-1. Previouswork

showed that an AmIII3 band at ∼1258 cm-1 indicates the pure

R-helical conformation, a band at∼1280 cm-1 indicates a π-helix(bulge), and a band at∼1240 cm-1 indicates a 310-helix (25). The

AmIII3 band in pure water narrows at higher temperatures as

previously observed (26, 27), indicating decreased concentra-

tions of 310-helix and π-bulge conformations relative to the pure

R-helix concentration as the temperature increases. The AmIII3band in 50% methanol shows a shoulder at ∼1258 cm-1 and

another shoulder at ∼1280 cm-1, while the ∼1240 cm-1 com-

ponent is missing, indicating a lack of 310-helices. All helix

spectra show an AmIII3 band at ∼1200 cm-1, indicating turn

structures (26). Calculated R-helix-like spectra in other alcohols

FIGURE 1: Temperature dependence of the CD spectra of 1 mg/mLAP in pure water.

FIGURE 2: (a) θ222 of AP in 25% (v/v) alcohol. (b) θ222 of AP in 50%(v/v) alcohol. (c) Δθ222 (θ222 in 50% alcohol minus θ222 in 25%alcohol).

3338 Biochemistry, Vol. 49, No. 15, 2010 Xiong and Asher

(not shown) are essentially identical to those in methanol,

indicating similar ensembles of helical conformations.We calculated the Gibbs free energy landscapes of AP

(Figure 7) along the Ψ folding coordinate from the UVRR byusing themethodology ofMikhonin et al. (19, 26, 28). The energylandscape (Figure 7) is bumpy within the R-helix-like basin.Within this basin, the pure R-helix conformation (Ψ ∼ -45�) isalways lowest in energy, followed by the π-bulge conformation.The 310-helix conformation (Ψ ∼ -20�) lies at a slightly higherrelative energy in pure water, but at much higher energies inalcohols. As the temperature increases, the R-helix basin Gibbsfree energy in pure water increases, indicating that the R-helix isdestabilized relative to the PPII-like conformation. The relativeR-helix basin energies change very little with temperature in 50%alcohols. For all temperatures, the lowest R-helix Gibbs free

FIGURE 3: 204 nm excited UVRS of AP in pure water (;) andAP in50% methanol (---) at 10 �C. The UVRS of AP in pure water wasscaled to facilitate comparison.

FIGURE 4: Temperature dependence of 204 nm excited UVRS of APin 50% methanol (;) and UVRS of AP in pure water at 2 �C (---).The methanol contribution was subtracted. The UVRS of AP wasscaled to facilitate comparison.

FIGURE 5: Raman-calculated AP R-helix-like fractions [primarilyR-, 310-, and π-helix (bulge)] of AP in different solutions.

FIGURE 6: Calculated R-helix-like spectra of AP in pure water (;)and in 50% (v/v) methanol (---). Calculated R-helix-like differencespectra were normalized to the intensity of the AmIII1 band.

Article Biochemistry, Vol. 49, No. 15, 2010 3339

energies occur in 50% TFE, followed by ethanol, methanol,and finally pure water. The same trend is seen with theπ-bulge energies. The alcohol-induced π-bulge energy decreaseis larger than that of the R-helix. Turn conformations arestabilized by alcohols, consistent with previous observations thatalcohols stabilize turns over PPII-like conformations (29). Incontrast, 310-helix conformations are dramatically destabilizedby alcohols.

DISCUSSION

Impact of Alcohols on the Gibbs Free Energies ofHelices. Numerous studies indicate that alcohols induce R-helixformation in proportion to the bulkiness of their alcohol hydro-carbon group (8, 12, 18, 30). This is confirmed by our UVRRresults which show that the R-helix has the lowest Gibbs freeenergy in 50% TFE, followed by ethanol and methanol.

Alcoholmolecules displace water in the peptide hydration shellwhich increases the hydrophobicity of the peptide-solvent inter-

face, which should enhance intramolecular hydrogen bondingwhich should increase the R-helical content (31, 32). Previousstudies (33) indicate that 310-helices allow greater solvent accessto the peptide bonds and thus are favored as the solventhydrophilicity increases. In contrast, the R-helix and π-helix aremore favored as the solvent hydrophobicity increases. It is alsoknown that the 310-helix is favored in the peptide terminal regionswhere solvent exposure is greatest (34).TFE Induces Multihelix Segments. Our UVRR measure-

ments that indicate that 50% TFE most stabilizes R-helix-likeconformations appear to conflict with the CD measurementswhich show that 50% TFE does not significantly stabilizeR-helical conformations more than 25% TFE. Previous stu-dies (22, 35-37) showed that UVRR-calculated R-helical con-formation concentrations are higher than those calculated fromCD (36) because the magnitude of the molar ellipticity perpeptide bond (PB) decreases dramatically as the number ofPBs within an R-helix decreases (11, 38, 39). In contrast, Ramanis more linear; each peptide bond independently contributes tothe Raman intensity (36, 40) [except for the AmI band of theR-helical conformation where strong coupling between AmIvibrations exists (41)]. Thus, we can explain the spectroscopicresults by proposing that TFE induces themostR-helical PBs butalso breaks long helices into short helices (see Appendix I).Recent studies have showed that TFE binds strongly to pep-tides (42, 43), while ethanol does not directly bind (10).

To quantify the dependence of the CDmolar ellipticity per PBof an R-helix, θn, on the number of PBs within the helix, n, wefitted our experimental data to the empirical equation proposedby Chen et al. (39) (see Appendix II):

θn ¼ - 34530 1-1:6

n

� �deg cm2 dmol- 1 ð1Þ

This allows us to relate the observed θ222 values to the UVRR-calculated helical fractions (see Appendix III). θ222 in 50% TFEcalculated in Figure 8 is modeled to be less negative than that in50% ethanol at low temperatures. [Calculated θ222 values areslightly more negative than those measured in Figure 2b becausethe NaClO4 used as an internal standard in the UVRR measure-ments but not included in the CD measurements stabilizes theR-helix conformation (27).]

FIGURE 7: Calculated Gibbs free energy landscape of AP along theRamachandran Ψ angle coordinate (blue line) in pure water, (greenline) in 50%methanol, (black line) in 50% ethanol, and (red line) in50% TFE. The PPII-like conformation is the reference state.

FIGURE 8: Calculatedθ222 ofAP inpurewater and in 50%(v/v) alcohols.

3340 Biochemistry, Vol. 49, No. 15, 2010 Xiong and Asher

CONCLUSIONS

CDandUVRRmeasurements indicate that TFEmost stabilizesthe R-helix, followed by ethanol, methanol, and pure water. Wedetermined the Gibbs free energy landscape from the UVRRspectra and found that the alcohol-inducedπ-bulge energy decreaseis larger than that of theR-helix, while the 310-helix energy increasesdue to the alcohol-increased hydrophobicity. Turns are stabilizedby alcohols as well. We also found that while TFE induces moreR-helices, it favors multiple, shorter helical segments.

ACKNOWLEDGMENT

WethankBhavyaSharma, LuMa,ZhenminHong, andDavidPunihaole for useful discussions.

APPENDIX I

TFE Induces More but Shorter Helices Than OtherAlcohols. Suppose θR(θr) is the mean residue ellipticity for thepure R-helix [melted conformation(s)] [θR = -26000 deg cm2

dmol-1; θr=-3500 deg cm2 dmol-1 (22)], θ is themeasuredmeanresidue ellipticity of AP, and fRRaman is the UVRR-calculatedconcentration of the R-helix-like conformation of AP. The CDR-helix fraction, fRCD, is calculated by (22)

fRCD ¼ θ- θrθR - θr

ð2Þ

fRRaman and fRCD are similar in pure water, 50% methanol, and50% ethanol. A significant difference between fRRaman and fRCD isobserved in 50% TFE which is likely due to the TFE-inducedformation of short helices that show weakened CD signals (36).The proportionality between fRRaman and fRCD is decreased in TFE(Figure 9) relative to that in ethanol and methanol, indicating thatTFE produces more but shorter helices. Given the energy cost ofnucleating helix segments (44-47), it is unlikely that the 21-residueAP adopts more than two helix segments.

APPENDIX II

Quantify theDependence of the CDMolar Ellipticity perPB of an R-Helix, θn, on the Number of PBs within theR-Helix, n. Suppose pi is the probability of AP containing asingle R-helix segment containing ni R-helical PBs, n is the

statistical average number of helical PBs, and N = 20, which isthe total number of AP PBs. Thus

θ ¼Xi

θnini

Nþ N- ni

Nθr

� �pi ð3Þ

Substituting the empirical equation proposed by Chen et al. (39),θn = θ¥(1 - k/n), into eq 3 yields

θ ¼Xi

θ¥ni

N-

k

N

� �þN- ni

Nθr

� �pi

¼ θ¥N

Xi

nipi -θ¥k

N

Xi

pi þ θrXi

pi -θrN

Xi

nipi

¼ θ¥N

n-θ¥k

Nþ θr -

θrN

n ð4Þ

As the Raman intensity is more linear (36, 40), the UVRR-calculated concentrations of R-helix-like conformations, fRRaman,more accurately monitor the fractions of R-helical PBs. Thus

fRRaman ¼ n

Nð5Þ

substituting eq 8 into eq , thus

θ ¼ ðθ¥ - θrÞfRRamanþ θr -k

Nθ¥

� �ð6Þ

We fit the experimental data to eq 6 (Figure 10) to obtain valuesof θn and n:

θn ¼ - 34530 1-1:6

n

� �deg cm2 dmol- 1 ð7Þ

APPENDIX III

Predicting CD Ellipticity from Raman-Calculated Con-centrations of R-Helix-like Conformations.Case 1. AP contains a single R-helix segment:

θ ¼ ðθ¥ - θrÞfRRamanþ θr -k

Nθ¥

� �ð8Þ

Case 2.APcontains two helix segments: pi is the probability ofAP containing one-helix segments of ni1 PBs and the second helixsegment of ni2 PBs; n1 and n2 are the average numbers of helical

FIGURE 9: fRCD vs fRRaman of AP.

FIGURE 10: Linear fit of previouslymeasuredθ222 and fRRaman valuesof AP in pure water (27).

Article Biochemistry, Vol. 49, No. 15, 2010 3341

PBs in the first and second helix segments, respectively (n1þ n2=NfRRaman). Thus

θ ¼Xi

θni1ni1

Nþ θni2

ni2

NþN- ni1 - ni2

Nθr

� �pi

¼Xi

θ¥ni1

N-

k

N

� �þ θ¥

ni2

N-

k

N

� �þN- ni1 - ni2

Nθr

� �pi

¼ θ¥N

Xi

ðni1 þ ni2Þpi - 2k

N

Xi

pi þ θrXi

pi -θrN

Xi

ðni1 þ ni2Þpi

¼ θ¥N

Xi

ðniÞpi - 2k

N

Xi

pi þ θrXi

pi -θrN

Xi

ðniÞpi

¼ θ¥N

ðn1 þ n2Þ- 2k

Nþ θr -

θrN

ðn1 þ n2Þ

¼ θ¥fRRaman - 2k

Nþ θr - θrfRRaman

\θ ¼ ðθ¥ - θrÞfRRamanþ θr -2k

Nθ¥

� �ð9Þ

We can then predict the observed CD ellipticity, θ, from fRRaman

in purewater, 50%methanol, and 50% ethanol by using eq 8 andin 50% TFE by using eq 7. The results are shown in Figure 8.

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